Many proteins, including actin, are targets for S-glutathionylation, the reversible formation of mixed disulphides between protein cysteinyl thiol groups and glutathione (GSH) that can be induced in cells by oxidative stress. Proposed mechanisms of protein S-glutathionylation follow mainly two distinct pathways. One route involves the initial oxidative modification of a reduced protein thiol to an activated protein, which may then react with GSH to the mixed disulphide. The second route involves the oxidative modification of GSH to an activated form such as glutathione disulphide (GSSG), which may then react with a reduced protein thiol, yielding the corresponding protein mixed disulphide. We show here that physiological levels of GSSG induce a little extent of actin S-glutathionylation. Instead, actin with the exposed cysteine thiol activated by diamide or 5,5'-dithiobis(2-nitrobenzoic acid) reacts with physiological levels of GSH, incorporating about 0.7 mol GSH/mol protein. Differently, an extremely high concentration of GSSG induces an increased level of S-glutathionylation that causes a 50% inhibition in actin polymerization not reversed by dithiotreitol. In mammalian cells, GSH is present in millimolar concentrations and is in about 100-fold excess over GSSG. The high concentration of GSSG required for obtaining a significant actin S-glutathionylation as well as attendant irreversible changes in protein functions make unlikely that actin may be S-glutathionylated by a thiol-disulphide exchange mechanism within the cell.
|Titolo:||Actin S-glutathionylation: evidence against a thiol-disulphide exchange mechanism|
|Parole Chiave:||Anti-GSH antibody ; reversed-phase HPLC ; glutathione disulphide ; protein mixed disulphides ; protein S-glutathionylation ; De-glutathionylation ; free radicals|
|Settore Scientifico Disciplinare:||Settore BIO/06 - Anatomia Comparata e Citologia|
|Data di pubblicazione:||2003|
|Digital Object Identifier (DOI):||10.1016/S0891-5849(03)00504-5|
|Appare nelle tipologie:||01 - Articolo su periodico|